ABSTRACT IRGM is a gene that regulates autophagy and has been identified as Crohn?s disease susceptibility gene. The proposed studies will define its role in intestinal immunity, which is currently unknown. This will allow a greater understanding of how some individuals acquire Crohn?s disease, as well as identify biochemical pathways that may be targeted for better therapeutics and/or diagnostics. Current dogma suggests that impaired autophagy, through lack of IRGM or other autophagy proteins, undermines intestinal immunity to enteric bacteria by reducing autophagic clearance of those bacteria. However, a distinct hypothesis proposed here is that lack of IRGM or a mouse homologue, Irgm1, leads to decreases in autophagic removal of mitochondria in certain intestinal immune cells. Accumulation of defective mitochondria perturbs cellular homeostasis, particularly in myeloid cells, reducing their ability to drive innate immune responses to enteric bacteria. This will be addressed with the following aims: Aim 1. Determine the immune components regulated by Irgm1 that are required for resistance to pathogenic enteric bacteria. Preliminary studies indicate that resistance to Citrobacter rodentium ? a mouse model of adherent invasive Escherichia coli linked to Crohn?s disease ? is markedly reduced in mice with a global knock-out of Irgm1. It is not clear which leukocyte populations in the lamina propria are impacted by Irgm1 deficiency. These studies will address the hypothesis that Irgm1 deficiency increases cell death in both macrophages and dendritic cells reducing their availability to drive a robust Th1 response. Conditional Irgm1- deficient mice will be used to identify the primary cell type(s) responsible for Irgm1-dependent susceptibility to C. rodentium and to determine how Irgm1 regulates homeostasis and function of those cells. Aim 2. Determine the mechanism(s) through which Irgm1 modulates myeloid cell function. These studies will address the hypothesis that the impaired autophagy that accompanies Irgm1-deficiency reduces autophagic removal of damaged mitochondria in myeloid cells. The consequential accumulation of damaged mitochondria, as well as the metabolic shifts caused by the damaged mitochondria, increase apoptotic cell death. Comprehensive analyses of macrophages and DC will be performed in vitro and in vivo, using pharmacologic and/or genetic tools to probe autophagy, mitochondrial pathways, metabolism, and cell death. Aim 3. Determine how disease variants of the IRGM gene alter human leukocyte function. The mechanism through which polymorphisms in human IRGM affect immune function and drive Crohn?s disease is unknown. These studies will address the hypothesis that - similar to mouse Irgm1 - perturbations in human IRGM expression alter autophagy, leading to changes in mitophagy and metabolism with downstream impacts on myeloid cell survival and function. This will be tested using leukocytes isolated from individuals carrying the IRGM major allele or disease risk allele, assessing autophagic, mitochondrial, metabolic, and functional outputs.